Recovery of a protein of interest from a culture broth may be hampered by the fact that a significant amount of the protein of interest is not in a solubilized form.
Said problem may occur when e.g. the protein of interest is bound to components in the sludge as such or due to the fact that a significant amount of the protein of interest is precipitated or crystallized prior to harvest from the culturebroth.
Sludge binding of a protein of interest denotes that the protein is bound to solids in the culture medium, such as cell solids or other solid components in the medium.
In relation to an effective recovery of the protein this may be a significant problem.
A number of methods have been applied for solving or minimizing said problem.
In many cases low or varying recovery yields have been accepted, in other cases the problem has been diminished through additions of e.g. ionic/nonionic surfactants, salts, anti/defoaming agents, alcohols, substrate or substrate analogs for the enzyme in question.
In many cases such techniques have been of low efficiency, in other cases complicated procedures have been developed (e.g. liquid-liquid separation systems) requiring large amounts of agent(s) added.
Some examples are found in the recovery of lipases, which are enzymes with a high tendency towards binding to fermentation solids and/or potential filter aids used in recovery.
WO 97/23604 describes a method for recovering of a lipase from a fermentation broth.
Essential steps in the described method comprise the use of both a nonionic surfactant and an alcohol so that a final composition is obtained which contains the microbial protein (preferably a lipase), the nonionic surfactant and the alcohol. See e.g. claim 1 of said WO 97/23604 document.
EP 0574050A1 describes a method for recovering of a hydrophobic product, preferably a lipolytic enzyme, (see column 3, line 47-50) from a fermentation broth. Essential steps in the described method comprise the use of both a nonionic surfactant and a salt in the process. See e.g. claim 1.
Journal of Biotechnology, 26 (1992) 111-142 is a review article describing the state of the art for different purification strategies of a lipase. On page 129 it is mentioned that lipase purification is generally hampered by the problem of solubilization of the lipase. The article summarizes the state of the art solutions to this problem which comprise very complex purification strategies, such as Liquid-liquid extraction, aqueous two-phase systems, specific membrane processes, and immunopurification.
For proteins precipitated or crystallized prior to harvest of broth, no real efficient methods exist today. Examples of solubilizing with large amounts of solubilization agents are typically tried (e.g. urea). Other examples are solid-solid separation techniques wherein the precipitated or crystallized protein in question is separated from the other solids in the broth,--such separations are often not possible and are always complicated.